Botanical Antioxidants

ABSTRACT

A botanical extract that exhibits antioxidant activity, wherein the botanical extract s at least an extract from the genus  Anacardium.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Patent ApplicationNo. 62/725,435, filed 31 Aug. 2018, the disclosure of which isincorporated herein in its entirety by reference.

BACKGROUND OF THE INVENTION

Field or the Invention. The present invention generally relates toinhibitors of oxidation-induced DNA damage, and more particularly tobotanical inhibitors of oxidation-induced DNA damage and the use of suchbotanical inhibitors as an antioxidant,

Oxygen is a highly reactive atom that is capable of becoming part ofpotentially damaging molecules called “free radicals”. Free radicals,commonly known as reactive oxygen species (‘ROS’), contain one or moreunpaired electrons in their outermost orbital. Common examples ofreactive oxygen species include peroxyl radical (ROO′), superoxide anion(O₂′), reactive hydroxyl (OH′), and hydrogen peroxide (H₂O₂) radicals.These free radicals are generated spontaneously in living organismsduring metabolism. As free radicals are highly unstable, they react withother molecules in their vicinity (e.g proteins, lipids, or DNA) toattain stability by taking electrons from those molecules, therebycausing damage to the cell and initiating a chain reaction offree-radical generation.

An imbalance between the generation of free radicals and cellularantioxidant can lead to oxidative stress. Oxidative stress occurs whenan oxygen molecule splits into single atoms with unpaired electrons,which are called free radicals. Since electrons prefer to be in pairs,these free radicals scavenge the body to seek out other electrons inwhich to pair with, causing damage to cells, proteins, and DNA in doingso. The term oxidative stress is used to describe the condition ofoxidative damage resulting when the critical balance between freeradical generation and antioxidant defenses is unfavorable.

Oxidative stress, arising as a result of an imbalance between freeradical production and antioxidant defenses, is associated with damageto a wide range of molecular species including lipids, proteins, andnucleic acids. An excess of oxidative stress can lead to the oxidationof lipids and proteins, which is associated with changes in theirstructure and functions. Short-term oxidative stress may occur intissues injured by trauma, infection, heat injury, hypertoxia, toxins,and excessive exercise. These injured tissues produce increased radicalgenerating enzymes (e.g., xanthine oxidase, lipogenase, cyclooxygenase)activation of phagocytes, release of free iron, copper ions, or adisruption of the electron transport chains of oxidativephosphorylation, producing excess ROS. Oxidative stress has beenimplicated in the etiology of several degenerative diseases, such asstroke, Parkinson's disease, Alzheimer's disease, rheumatoid arthritis,diabetes mellitus, peptic ulcer, gene mutations and cancer, heart andblood disorders, and inflammatory diseases. Oxidative stress is nowthought to make a significant contribution to all inflammatory diseases(arthritis, vasculitis, glomerulonephritis, lupus erythematous, adultrespiratory diseases syndrome), ischemic diseases (heart diseases,stroke, intestinal ischema), hernochromatosis, acquired immunodeficiencysyndrome, emphysema, organ transplantation, gastric ulcers, hypertensionand preeclampsia, neurological disorder (Alzheimer's disease,Parkinson's disease, muscular dystrophy), alcoholism, smoking-relateddiseases, and many others.

Antioxidants are capable of stabilizing, or deactivating, free radicalsbefore they attack cells. Application of external source of antioxidantscan assist in coping with oxidative stress. These include syntheticantioxidants such as butylated hydroxytoluene and butylatedhydroxyanisole; however, these synthetic antioxidants have recently beenreported to be dangerous for human health. Thus, the search foreffective, nontoxic, natural compounds with antioxidative activity hasbeen intensified in recent years.

Antioxidants are reducing agents, examples of which includenutrient-derived antioxidants such as ascorbic acid (Vitamin C),tocopherols and tocotrienols (Vitamin E), carotenoids, and poly phenols;antioxidant enzymes such as superoxide dismutase, glutathioneperoxidase, and glutathione reductase; metal binding proteins such asferritin, lactoferrin, albumin, and ceruloplasimin; and trace metals(e.g., zinc and molybdenum). These antioxidants can scavenge reactiveoxygen species and inhibit the chain reaction by donating an electron tothe free radical. The antioxidant defense system, supported by dietaryantioxidants, protects the body from free radicals. However, duringoxidative stress, antioxidants are insufficient to maintain homeostasis.In such instances, antioxidants can be given as supplements, theconsumption of which can significantly reduce the risk for freeradical-associated diseases.

Phytomedicine plays an important role in the management of most of thesediseases, with plants being a potential source of natural antioxidants.Studies have shown that the consumption of polyphenolic compounds foundin tea, fruits, and vegetables is associated with low risk of thesediseases. Consequently, there is a growing research interest in plantsthat contain antioxidants and health-promoting phytoconstituents aspotential therapeutic agents. Medicinal plants provide a safe,cost-effective, ecological alternative to Chemical antioxidants, whichcan be toxic on prolonged exposure.

The cashew tree (Anacardium occidentale Linn) is originally from theAmazon, and has subsequently been transplanted to India, Eastern Africa,and other countries for cultivation. The tree produces a very peculiarapple or fruit in the form of a swollen peduncle. Externally at the endof this peduncle the cashew nut grows in its own grey coloredkidney-shaped hard shell. This shell has a soft leathery outer skin anda thin hard inner skin referred to as the husk or testa, which surroundsthe kernel. Between these two skins is a honeycomb structure containingthe cashew nut shell liquid. This liquid comprises anacardic acid,cardanol, and cardol among other ingredients. Anacardic acid is asalicylic acid, while cardanol and cardol are substituted phenols.

The various parts of the fruit have been studied for their uses, inaddition to being an edible food, the juice from the cashew apple isused in beverages, while the fruit extract has shown benefit in weightmanagement. Cashew nut shell liquid has been extracted for variousindustrial and agricultural applications, including friction linings,paints, laminating resins, rubber compounding resins, cashew cements,polyurethane based polymers, surfactants, epoxy resins, foundrychemicals, chemical intermediates, insecticides, and fungicides. Cashewtesta has been used in tanning materials.

As part of a healthy lifestyle and a well-balanced, wholesome diet,antioxidant supplementation is recognized as an important means ofimproving free radical protection. As noted above, there is a need foreffective, nontoxic, natural compounds with antioxidant activity. Thepresent invention provides one such solution.

BRIEF SUMMARY OF THE INVENTION

Disclosed herein is a botanical extract comprising catechins, whereinthe extract has been standardized to a catechin content of about 15.0w/w % or greater, based on total weight of the extract, wherein thebotanical extract exhibits antioxidant activity, and wherein thebotanical extract comprises at least an extract from the genusAnacardium. In one aspect, the botanical extract can be obtained from aplant chosen from Anacardium humile, Anacardium othonianum, Anacardiumgiganteum, Anacardium nanum, Anacardium negrense and/or Anacardiumoccidentale. Preferably, the botanical extract is at least fromAnacardium occidentale L. In one embodiment, the botanical extract isfrom at least the testa of the fruit of Anacardium occidentale L.

In another aspect, the present invention provides a compositioncontaining the botanical extract of the testa of Anacardium occidentaleL., wherein the botanical extract exhibits anti-oxidant activity. Thebotanical extract can be present in the composition in an amount ofabout 4.0 μg/mL or greater. For example, the botanical extract can bepresent in the composition in an amount of about 4.0 μg/mL to about2000.0 μg/mL.

In one aspect, the composition containing the botanical extract of thecashew testa inhibits γ-H2AX activity. In one embodiment, the botanicalextract is present in the composition in an amount of about 4.0 μg/mL toabout 2000.0 μg/mL. Compositions can include, for example, food and/orbeverage compositions infused with the extract.

Also provided herein is a dietary supplement having antioxidantproperties, wherein the supplement comprises a cashew testa extract in atherapeutically effective amount. For example, the cashew testa extractcan be present in the dietary supplement in an amount of about 4.0μg/mL, to about 2000.0 μg/mL.

The present invention further provides a method of inhibitingoxidation-induced DNA damage in a subject by administering a compositioncomprising the botanical extract of the testa of Anacardium occidentaleL. at a concentration of about 4.0 μg/mL to about 2000.0 μg/mL.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is an HPLC chromatogram of cashew testa extract at 275 nm over aretention time of from 0 minutes (start) to 20 minutes.

FIG. 2 is LC/MS and LC/PDA (wavelengths of 280 and 350 nm) chromatogramsof cashew testa extract.

FIG. 3 is a graph illustrating the efficacy of cashew testa extract ininhibiting DNA damage compared to catechin and soliprin standards.

FIG. 4 is a graph illustrating cell viability of a cell culture whendosed with various doses of cashew testa extract versus catechin andsoliprin standards.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the surprising discovery that thetesta of the cashew (Anacardium) is substantially high in certainflavonoids. In particularly, it has been discovered that the extract ofcashew testa comprises catechin and epicatechin as major components, aswell as procyanidins. Data noted herein demonstrates that cashew testaextract is beneficial in protecting DNA from oxidative stress-induceddamage.

For the present application, the term “composition” refers to a productthat treats, improves, promotes, increases, manages, controls,maintains, optimizes, modifies, reduces, inhibits, or prevents aparticular condition associated with a natural state, biological processor disease or disorder. For example, a composition improves theinhibition of oxidation and/or reduces inflammation, and the like in asubject. The term composition includes, but is not limited to,pharmaceutical (i.e., drug), over-the counter (OTC), cosmetic, food,food ingredient or dietary supplement compositions that include aneffective amount of an extract, at least one component thereof, or amixture thereof. Exemplary compositions include cream, cosmetic lotion,pack or powder, or as an emulsion, lotion, liniment foam, tablets,plasters, granules, or ointment. Compositions can also includebeverages, for example, beverages infused with an effective amount of anextract, or a tea satchel containing an effective amount of an extract.Non-limiting examples of food compositions containing an effectiveamount of an extract include baked goods, protein powders, meatproducts, dairy products, and confectionary.

As used herein, the term “extract” or “botanical extract” refers to asolid, viscid, or liquid substance or preparation that includes one ormore active ingredients of a substance of at least the plant Anneardium(e.g., Anacardium humile, Anacardium othonianum, Anacardium giganteum,Anacardium nanum, Anacardium negrense, and/or Anacardium occidentale),preferably Anacardium occidentale L. Preferably, the active ingredientis derived from the extract of the testa of the cashew. The extract canbe prepared using a solvent such as water, lower alcohols of 1 to 4carbon atoms (e.g., methanol, ethanol, butanol, etc.), ethylene,acetone, hexane, ether, chloroform, ethylacetate, butylacetate,dichloromethane, N,N-dimethylformamide (‘DMF’), dimethylsulfoxide(‘DMSO’), 1,3-butylene glycol, propylene glycol, and combinationsthereof, hut also a fraction of the crude extract in such a solvent. Solong as it assures the extraction and preservation of the activeingredient(s), any extraction method may be employed.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” of a pure compound, composition, extract, extractmixture, component of the extract, and/or active agent or ingredient, ora combination thereof refers to an amount effective at dosages and forperiods of time sufficient to achieve a desired result. For example, the“effective amount” or “therapeutically effective amount” refers to thatamount of a pure compound, composition, extract, botanical extract,extract mixture, botanical extract mixture, component of the extract,and/or active agent or ingredient, or a combination thereof of thisinvention which, when administered to a subject (e.g., mammal, such as ahuman), is sufficient to effect treatment, such as improving theinhibition of oxidation and/or reducing inflammation, and the like in asubject. The amount of a composition, extract, botanical extract,extract mixture, botanical extract mixture, component of the extract,and/or active agent or ingredient of this disclosure that constitutes an“effective amount” or “therapeutically effective treatment” will varydepending on the active agent or the compound, the condition beingtreated and its severity, the manner of administration, the duration oftreatment, or the age of the subject to be treated, but can bedetermined routinely by one of ordinary skill in the art having regardto his own knowledge and to this disclosure.

The term “pharmaceutically acceptable” means those drugs, medicaments,extracts or inert ingredients, which are suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,incompatibility, instability, irritation, and the like, commensuratewith a reasonable benefit/risk ratio.

The terms “administer”, “administered” “administers”, and “administeringare defined as providing a composition to a subject via a route known inthe art, including but not limited to intravenous, intra-arterial, oral,parenteral, buccal, topical, transdermal, rectal, intramuscular,subcutaneous, intraosseous, transmucosal, or intraperitoneal routes ofadministration. In preferred embodiments, oral routes of administering acomposition are suitable.

As used herein, the term “subject” or “individual” includes mammals towhich a composition may be administered. Non-limiting examples ofmammals include humans, non-human primates, canines, felines, equines,bovines, rodents (including transgenic and non-transgenic mice) or thelike. In some embodiments, the subject is a non-human mammal, and insome embodiments, the subject is human.

As used herein, the term “carrier” refers to a composition that aids inmaintaining one or more plant extracts in a soluble and homogeneousstate in a form suitable for administration, which is nontoxic and whichdoes not interact with other components in a deleterious manner.

Unless indicated otherwise, all proportions and percentages recitedthroughout this disclosure are by weight.

The present invention provides a plant-based inhibitor capable ofinhibiting DNA damage due to oxidative stress. More particularly, thepresent invention is directed towards a botanical extract of the cashewtesta from the genus Anacardium. Such botanical extracts have been foundto be capable of inhibiting oxidative stress-induced DNA damage byneutralizing free radicals, thereby terminating the chain reactioncreated by the free radicals. By terminating the chain reaction, damagedue to the free radicals by their reaction with important macromoleculessuch as DNA, protein, lipids, or the cell membrane is prevented orinhibited.

Useful botanical extracts capable of inhibiting DNA damage due tooxidative stress according to the present invention include botanicalextracts from the genus Anacardium. More particularly, the plant-basedinhibitor is a botanical extract chosen from one or more of the speciesAnacardium humile, Anacardium othonianum, Anacardium giganteum,Anacardium nanum, Anacardium negrense, and/or Anacardium occidentale.Preferably, the botanical extract is from the species Anacardiumoccidentale. In one embodiment, the botanical extract is from the testaof the species Anacardium occidentale.

Compositions capable of inhibiting DNA damage due to oxidative stressaccording to the present invention may include one or more compoundsthat may function as active ingredients. The compound may be a componentof the botanical extract. For example, the compound can be aphytochemical present in the plant from which the plant extract isobtained. The compound may be at least partially responsible forinhibiting DNA damage due to oxidative stress. The compound can be anycompound capable of inhibiting DNA damage due to oxidative stress. Inone embodiment, the compound is chosen from the phytochemicalscatechins, epicatechins, and/or procyanidins (e.g., A, B, trimer,tetramer).

Generally, one or more parts of a plant can be used to produce a plantextract including, but not limited to, the root, the stem, the leaf, theflower, the fruit, the seed, and the testa of the seed. In the presentinvention, at least the testa of the seed is used—alone or with otherplant parts—to produce the plant extract. The testa from the Anacardiumplant can be commercially obtained from various sources. The extract ofthe cashew testa can be obtained using any suitable extractiontechnique.

In this regard, one or more parts of the plant, particularly the testaof the plant, can be collected and milled. Thereafter, the milledmaterial can be extracted using a suitable solvent. The solvent can beremoved in a concentration step. For example, the extracted material canhe screened or filtered to create a supernatant and a cake. The cake canbe pressed to remove a substantial portion of the liquid, which can beadded to the supernatant. The cake can then be dehydrated and used as afiber source. The supernatant can be distilled to remove the solvent ora portion thereof, to form a plant extract liquid concentrate. Theremoved solvent can be recycled. The concentrate can be dried (e.g., byspray drying) to provide a dried plant extract. This dried plant extractcan be assayed and/or standardized as described herein. Preferably, thedried plant extract is derived from Anacardium occidentale, particularlythe testa of the plant Anacardium occidentale.

Suitable solvents for the extraction process include water, alcohol, ormixtures thereof. Exemplary alcoholic solvents include, but are notlimited to, C₁-C₇ alcohols (e.g., methanol, ethanol, propanol,isopropanol, and butanol), hydro-alcohols or mixtures of alcohol andwater (e.g., hydroethanol), polyhydric alcohols (e.g., propylene glycoland butylene glycol), and fatty alcohols. Any of these alcoholicsolvents can be used in the form of a mixture. In one embodiment, theplant extract is extracted using ethanol, water, or a combinationthereof (e.g., a mixture of about 70% ethanol and about 30% water). Inanother embodiment, the plant extract is extracted using only water.

In one embodiment, the plant extract can be obtained using an organicsolvent extraction technique, in another embodiment, solvent sequentialfractionation can be used to obtain the plant extract. Totalhydro-ethanolic extraction techniques can also be used to obtain theplant extract. Generally, this is referred to as a lump-sum extraction.

Total ethanol extraction can also be used. This technique uses ethanolas the solvent. This extraction technique can generate a plant extracthaving fat soluble and/or lipophilic compounds in addition to watersoluble compounds.

Another example of an extraction technique that can be used to obtainthe plant extract is supercritical fluid carbon dioxide extraction(‘SFE’). In this extraction procedure, the material to he extracted maynot be exposed to any organic solvents. Rather, carbon dioxide can beused as the extraction solvent—with or without a modifier—insuper-critical conditions (>31.3° C. and >73.8 bar). Those skilled inthe art will appreciate that temperature and pressure conditions can bevaried to obtain the best yield of extract. This technique can generatean extract of fat soluble and/or lipophilic compounds, similar to atotal hexane and ethyl acetate extraction technique.

The plant extract generated in the process can include a broad varietyof phytochemicals present in the extracted material. The phytochemicalscan be fat soluble or water soluble. Following collection of the extractsolution, the solvent can be evaporated, resulting in the extract.

The plant extract can be standardized to specified amount of aparticular compound. For example, the plant extract can be standardizedto a specified amount of an active ingredient or phytochemical presentin the extract. In one embodiment, the plant extract is standardized toa catechin content of about 15.0 wt % or greater, based on total weightof the extract.

The amount of plant extract present in the oxidative stress-induced DNAdamage inhibiting composition can depend upon several factors, includingthe desired level of oxidative stress-induced DNA damage inhibition, theoxidative stress-induced DNA damage inhibiting level of a particularplant extract or component thereof, and other factors. Preferably, theplant extract is present in an amount of from about 0.005 wt % orgreater, for example, from about 0.005 wt % to about 50.00 wt %, basedon total weight of the composition.

The oxidative stress-induced DNA damage inhibiting composition caninclude one or more acceptable carriers. The carrier can aid in enablingincorporation of the plant extract into an oxidative stress-induced DNAdamage inhibiting composition having a suitable form for administrationto a subject. A wide number of acceptable carriers are known in the art,and the carrier can be any suitable carrier. The carrier is preferablesuitable for administration to animals, including humans, and can beable to act as a carrier without substantially affecting the desiredactivity of the plant extract and/or any active ingredient. The carriercan be chosen based upon the desired administration route and dosageform of the composition.

Suitable dosage forms include liquid and solid forms. In one embodiment,the composition is in the form of a gel, a syrup, a slurry, or asuspension. In another embodiment, the composition is in a liquid dosageform such as a drink shot or a liquid concentrate. In a furtherembodiment, the composition is present in a solid dosage form, such as atablet, a pill, a capsule, a dragée, or a powder. When in liquid orsolid dosage form, the composition can be in a food delivery formsuitable for incorporation into food for delivery. Examples of suitablecarriers for use in solid forms (particularly tablet and capsule forms)include, but are not limited to, organic and inorganic inert carriermaterials such as gelatin, starch, magnesium stearate, talc, gums,silicon dioxide, stearic acid, cellulose, and the like. The carrier canbe substantially inert.

As an example, silicified microcrystalline cellulose can be used as acarrier or binder. Silicified microcrystalline cellulose is a physicalmixture of microcrystalline cellulose and colloidal silicon dioxide. Onesuch suitable form of silicified microcrystalline cellulose is ProSolySMCC® 90, available from Penwest Pharmaceutical Co., Patterson, N.J.Silicon dioxide, in addition to that provided by the silicifiedmicrocrystalline cellulose, may be added to the composition as aprocessing aid. For example, silicon dioxide can be included as aglidant to improve the flow of powder during compression in themanufacturing of solid dosage units, such as tablet.

In another embodiment, the carrier is at least a functional carrier suchas buckwheat or spelt. By the addition of functional carriers into thecomposition, additional benefits may be provided such as lower glycemicindex compared to standard carriers such as those mentioned above.Further, functional carriers can be allergan free (e.g., buckwheat), andby adding them into the production process, the botanical extracts ofthe invention may benefit from the flavonoids of these functionalcarriers, such as rutin and quercetin. Further, the high fiber contentof these functional carriers may also facilitate and regulate intestinaltransit. Finally, the added mineral benefit of selenium found in speltmay aid in metabolism.

The oxidative stress-induced DNA damage inhibiting composition caninclude other inert ingredients, such as lubricants and/or glidants.Lubricants aid in the handling of tablets during manufacturing, such asduring ejection from dies. Glidants improve powder flow during tabletcompression. Stearic acid is an example of an acceptablelubricant/glidant.

The oxidative stress-induced DNA damage inhibiting composition can bemade in solid dosage form, such as tablets and capsules. This formprovides a product that can be easily transported by an individual to aplace of eating, such as a restaurant, and taken prior to, during, orafter consumption of a foodstuff. The composition can be formulated intodosage units containing suitable amounts of the plant extract and/oractive ingredient that permit an individual to determine an appropriatenumber of units to take based upon appropriate parameters, such as bodyweight, foodstuff size, or carbohydrate (e.g., sugar) content.

In one embodiment, the botanical extract is present in the compositionin a therapeutically effective amount, such as an amount of about 4μg/mL or greater, preferably from about 4.0 μg/mL to about 2000.0 μg/mL,more preferably from about 15.0 μg/mL to about 1000.0 μg/mL, even morepreferably from about 30.0 μg/mL to about 500.0 μg/mL. The compositioncan be administered as a single dose, or in multiple doses. In oneexample, the compound is administered in up to three doses per day. Forexample, the compound may be administered prior to a meal, during ameal, or after a meal. In one embodiment, the composition is a dietarysupplement having antioxidant properties containing cashew testa extractin a therapeutically effective amount.

The dosage can be chosen to provide a level of inhibitory effect in asingle unit that may be effective for some individuals and/or somefoodstuffs, while also allowing for relatively simple dosage increasesto provide other levels of inhibitory effects that can be effective forother individuals and/or other foodstuffs.

The inhibiting composition can be in a form adapted for oral ingestion.This form can be configured as a single dosage form intended to providea specified dose of the plant extract. For example, the single dosageform can be a powder, a pill, a tablet, a capsule, or a drink shot. Thesingle dosage form can include, for example, from about 4.0 μg/mL toabout 2000.0 μg/mL of the plant extract.

EXAMPLES Examples—Materials and Chemical Profiling Example 1—Preparationof Cashew Testa Extract using 70% Ethanol Solvent

Dried cashew testa powder (Anacardium occidentale) (60 g) was loadedinto three 100 ml stainless steel tubes and extracted twice using asolvent of 70% ethanol in DI water with a Thermo Scientific™ Dionex™ ASE350 Accelerated Solvent Extractor at a temperature of 80° C. andpressure of 1500 psi. The extract solution was filtered and collected.The combined ethanol extract solution was evaporated with a rotaryevaporator under vacuum to give a crude cashew testa extract.

The extraction results are provided in the following Table 1—

TABLE 1 Extraction of cashew testa Extraction Yield Plant Part PlantPowder (g) Extract Weight (g) (wt %) Testa 60 23.78 39.63%

Example 2—Catechin Quantification of Cashew Testa Extract

Free catechins present in the cashew testa extract were determined usinga C18 reversed-phase column (Luna® 5 μm C18(2) 100 Å LC Column 250×4.6mm, available from Phenomenex®, Torrance, Calif., US) together with anHitachi high performance liquid chromatograph with photodiode arraydetector (‘HPLC/PDA’). For mobile phase A, the solvent was 0.10%phosphoric acid (‘H₃PO₄’) in water, and for mobile phase B, the solvent.B was acetonitrile (‘ACN’), which was used for elution at a flow ratedof 1.0 ml/min with UV absorbance at 275 nm and a column temperature of35° C. Catechin reference standards used were from Sigma-Aldrich Co. Thereference standards were dissolved in methanol (‘MeOH’): 0.1% H₃PO₄ (1:1ratio) with catechin (C1251) at a concentration of 0.5 mg/ml andepicatechin (E1753) at 0.1 mg/ml. Testing samples were prepared at 2mg/ml in 50% MeOH in 0.1% H₃PO₄ in a volumetric flask and sonicateduntil dissolved (approximately 10 minutes), and then cooled to roomtemperature, mixed well, and filtered through a 0.45 μm nylon syringefilter. HPLC analysis was performed by injecting a 20 μl sample into theHPLC. Table 2 below provides the gradient table of HPLC analyticalmethod—

TABLE 2 Gradient Table of HPLC Analytical Method Time (min) Mobile PhaseA Mobile Phase B 0.0 85.0 15.0 7.0 85.0 15.0 12.0 10.0 90.0 16.5 10.090.0 16.6 85.0 15.0 24.0 85.0 15.0

HPLC Catechin quantification results in cashew testa extract provided acatechin content of 9.40% and an epicatechin content of 6.12%, for atotal catechin content of 15.52% by weight, based on total weight of theextract. Accordingly, the cashew testa extract can be standardized to atotal catechin content of about 15.00% or greater by weight, based ontotal weight of the extract. The HPLC chromatogram for cashew testaextract at 275 nm wavelength is provided in FIG. 1.

Example 3—Chemistry Profiling of Cashew Testa Extract

Flavonoid compounds present in the cashew testa extract were determinedusing ultra high pressure liquid chromatography (‘HPLC’) and massspectrometry (ACQUITY® UPLC I-Class and XEVO® GS-XT-QT of system, bothavailable from Water Corporation, Milford, Mass. USA). The column usedwas an ACQUITY® UPLC HSS T3 2.1×100 mm, 1.8 μm, with a columntemperature of 40° C. and a sample temperature of 15° C. For the mobilephase, Solvent A was 10% acetonitrile (‘ACN’) in water (0.1% FormicAcid), and Solvent B was ACN. The acquisition range was 100-1500 Daltons(‘Da’), and the acquisition mode was electrospray ionization (‘ESI’)(−). Table 3 below provides the HPLC conditions—

TABLE 3 HPLC conditions for analyzing cashew testa extract Run Time(min) Injection Volume (μL) Concentration 20.00 2.00 1 mg/mL

Peak identification was based on accurate mass only. Digalloyl catechin,catechin and epicatechin were identified as the major components forcashew testa extract. Procyanidins were detected in the extract as well,including A- and B-type procyanidins, procyanidin tetramer, andprocyanidim trimer, with B-type procyanidins being the major componentof the procyanidins. Compounds identified, in addition to those justmentioned, included digalloyl catechin, vaccihein A,6″-p-coumaroylprunin, and dunalianoside B, among others. LC/MS andLC/PDA chromatograms of cashew testa extract obtained from the analysisare illustrated in FIG. 2.

Examples—Bioassay

Extracts of cashew testa were prepared with food-grade ethanol, and thenfiltered and dried as described above. Research grade reagents were usedfor the rest of the assay preparations. Extracts were dissolved indimethyl sulfoxide (‘DMSO ’) to a final concentration of 50 mg/mL, andthen diluted in appropriate buffer for each bioassay to workingconcentrations.

Example 4—DNA Damage Assay

When DNA damage—whether endogenous or exogenous—forms double strandedbreaks (‘DSBs’), it is always followed by phosphorylation of the histoneH2AX. H2AX is a variant of the H2A protein family, which is a componentof the historic octomer nucleosomes. It is phosphorylated by kinasessuch as ataxia telangiectasia mutated (‘ATM’) and ATM-Rad3-related(‘ATR’) in the PI 3K pathway. The protein γ-H2AX is the first step inrecruiting and localizing DNA repair proteins. DSBs can be induced bymechanisms such as ionizing radiation or cytotoxic agents andsubsequently, γ-H2AX foci quickly form. These foci represent the DSBs ina 1:1 manner and can be used as a biomarker for damage. An antibody canbe raised against γ-H2AX, which can therefore be visualized byimmunofluorescence through secondary antibodies The detection andvisualization of γ-H2AX by flow cytometry allow the assessment of DNAdamage, related DNA damage proteins, and DNA repair.

Human skin fibroblasts were seeded at a density of 8000 cells/well in96-well tissue culture plates. After 24 hours, cells were treated withtest compounds for 48 hours, after which the cells were treated with 1mM hydrogen peroxide for 4 hours to induce DNA damage. Hydrogen peroxideinduces DNA damage by creating breaks in the DNA. Cells were then fixedand stained with an antibody to the biomarker γ-H2AX, which is aphosphorylated histone variant that is found at double-strand breaks. Atsites of double-strand breaks, the histones are phosphorylated,indicating that the DNA is damaged and requires repair. An antibody forγ-H2AX—the phosphorylated historic variant—is useful in identifyingsites of DNA damage. Nuclei were stained with4′,6-diamidino-2-phenylindole (‘DAPI’), which is a fluorescent stainthat hinds to DNA. Pictures were taken with Image Xpress and analyzedwith Meta Xpress to measure fluorescence intensity in each conditiondivided by the total number of cells.

DNA damage in response to hydrogen peroxide treatment was measured bythe amount of γ-H2AX present in cells. Cashew testa extract was weretested for DNA damage inhibition at 50 and. 100 μg/mL based on theamount of γ-H2AX detected after treatment with hydrogen peroxide toinduce DNA damage. Catechin and soliprin at 25 μg/mL were used aspositive controls. As shown in FIG. 3, cashew testa extract at 100 μg/mLexhibited significant protection of DNA from damage in response tohydrogen peroxide treatment. Percent inhibition was calculated relativeto wells that were not treated with extracts but were exposed tohydrogen peroxide. From this Example, it is seen that pre-treatingfibroblasts with cashew testa extract at 100 μg/mL significantly reducedthe amount of γ-H2AX detected, showing the ability of cashew testaextract to protect DNA from oxidative stress-induced damage.

Cell Counting Kit-8 (‘CCK-8’) was used to determine the percentage ofviable cells in each treatment relative to an untreated control. CCK.-8reagent was added to the media (final concentration 10% total volume)and incubated for one (1) hour at 37° C. and 5% CO₂ before absorbancewas read on a Multimode Reader at 460 rim to determine the number ofviable cells relative to untreated control wells. The cashew testaextract treatment was not statistically significantly different from theuntreated controls (FIG. 4) and therefore not toxic to human skinfibroblasts.

The above data illustrates that the botanical extract of the testa ofAnacardium occidentale L has one or more compounds that exhibitanti-oxidant activity. More particularly, the cashew testa extract mayhave reasonable activities in ameliorating y-H2AX activity.

The above description discloses several methods and materials of thepresent invention. This invention is susceptible to modifications in themethods and materials, as well as alterations in the fabrication methodsand equipment. Such modifications will become apparent to those skilledin the art from a consideration of this disclosure or practice of theinvention disclosed herein. Further, unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs. Consequently it is not intended that this inventionbe limited to the specific embodiments disclosed herein, but that itcover all modifications and alternatives coming within the true scopeand spirit of the invention as embodied in the attached claims.

1. A composition comprising the botanical extract of the testa ofAnacardium occidentale L., wherein the botanical extract inhibits γ-H2AXactivity.
 2. The composition according to claim 1, wherein the botanicalextract is present in an amount of about 4.0 μg/mL or greater.
 3. Thecomposition according to claim 2, wherein the botanical extract ispresent in an amount of about 4.0 μg/mL to about 2000.0 μg/mL.
 4. Adietary supplement having antioxidant properties comprising a cashewtesta extract in a therapeutically effective amount, wherein thebotanical extract inhibits }-H2AX activity.
 5. The dietary supplementaccording to claim 4 wherein the cashew testa extract is present in anamount of about 4.0 μg/mL or greater.
 6. A botanical extract comprisingcatechins, wherein the extract has been standardized to a catechincontent of about 15.0 w/w % or greater, based on total weight of theextract, wherein the botanical extract exhibits antioxidant activity,and wherein the botanical extract comprises at least an extract from thegenus Anacardium.
 7. The botanical extract according to claim 6, whereinthe extract from the genus Anacardium is at least an extract fromAnacardium occidentale L.
 8. The botanical extract according to claim 7,wherein the extract from Anacardium occidentale L is from at least thetesta of the seed of Anacardium occidentale L.
 9. A method of inhibitingoxidation-induced DNA damage in a subject comprising administering acomposition comprising the botanical extract of the testa of Anacardiumoccidentale L at a concentration of about 4.0 μg/mL to about 2000.0μg/mL.